WO2007120034A1 - Method for rapid detection of lymphatic filariasis - Google Patents
Method for rapid detection of lymphatic filariasis Download PDFInfo
- Publication number
- WO2007120034A1 WO2007120034A1 PCT/MY2007/000021 MY2007000021W WO2007120034A1 WO 2007120034 A1 WO2007120034 A1 WO 2007120034A1 MY 2007000021 W MY2007000021 W MY 2007000021W WO 2007120034 A1 WO2007120034 A1 WO 2007120034A1
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- WIPO (PCT)
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- sxp
- detection
- chromatographic element
- chromatographic
- reaction zone
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Definitions
- the present invention relates to a method for rapid detection of lymphatic filariasis, particularly a method that detects anti-filarial IgG4 antibodies in a biological sample using a SXP/SXP-1 recombinant antigen and the technique of immunochromat ⁇ graphy.
- Lymphatic filariasis is a- parasitic and infectious tropical disease caused by a number of slender and thread-like parasitic filarial worms which invade blood circulation, lymphatics, lymph nodes and • other parts of the human body.
- lymphatic filarial parasites One hundred and ' twenty million people in 83 countries of the world are infected with lymphatic filarial parasites, and it is estimated that more than
- Lymphatic filariasis is transmitted to man by mosquitoes, -which introduce a large number of infective larvae into human.
- the female worijis produce microfilariae, which make their way i_o blood circulation and are taken up by a suitable mosquito species.
- the microfilariae first stage larvae, Li larvae
- the microfilariae undergo several moultings to form infective larvae (L 3 larvae)', which reach the blood circulation of the definite host through wound made by the bites of the mosquitoes .
- the infective larvae enter into the lymphatic system where they slowly mature into adult male and female worms.
- the male and 'female adult mate- to produce microfilariae which find their way to peripheral blood ' circulation.
- lymphatic filariasis The clinical manifestations of lymphatic filariasis can be divided into three types : 1. Asymptomatic: these individuals are outwardly non-symptomatic, but will demonstrate lymphatics and/or renal damage if appropriately tested.-
- Acute manifestations include "filarial fever” or acute attacks associated with inflammation of the lymphatic flodes and channels .
- the traditional or routine method to diagnose filarial infection depended on the direct demonstration of the microfilariae in blood using relatively cumbersome techniques and having to take into account the periodicity of microfi iariae in blood.
- This traditional method severe iy lacks sensitivity (25% - 40% sensitive) , thus missing many positive cases. This is due to the inability of the method to detect cryptic infections (before microfilariae are produced and after microfilariae ceased to be produced) , single sex infections, occult infections and low levels of microfilariae.
- Blood concentration techniques such as the Knoti ' s method and membrane filtration increases the sensitivity of detection but are usually not performed because they require ' venous blood taking. Polymerase chaia reaction
- SXP or SXP-I gene (Genbank accession No. M98813) was previously identified by imtmmoscreening of Brugia ma.la.yl cDNA library with immune sera from microfilariae positive patients with brugian and brancroftian filariasis (Dissanayake S., Xu M., Piessens WF. A cloned antigen 5 for serological diagnostic of Wuchereria hancrofti microfilariae with daytime blood samples. MoI B ⁇ ochem pa.ra.sito! 1992: 256-26) .
- the Brugia malayi-derived recombinant protein (JBm-SXP-I) was reported to be successful in identifying 83% (64/72) of bancroftian filariasis patients when tested with IgG4 -ELISA (Chandrashekar R., et al Molecular cloning of • Brugia malayi antigens for diagnosis of lymphatic filariasis. MoI Biochem Parasitol 1994:64 (2) : 262-271). With this method
- SXP/SXP-1 gene Although the expression products of SXP/SXP-1 gene has been reported over the years and proven . to successfully detect lymphatic filariasis in. infected patients, the SXP/SXP-1 gene was applied for the development of tests using ELISA format, . which requires several hours to perform. There is no report of the application of the expression products of
- Another object of the present invention is ' to provide a simple and ' rapid diagnostic kit employing the method outlined- above that can be performed by untrained personnel in a minimum amount of time.
- a method for rapid detection of lymphatic filariasis in a biological sample comprising the steps of: - a) Adding a buffer to reconstitute dified mouse monoclonal anti-human IgG4 antibody conjugated to a detection reagent prepared separately in a mierowell; b) Adding a biological sample to the sample receiving end of a chromatographic element; c) Allowing the biological sample to flow from the sample receiving end to the reaction zo ⁇ e of the chromatographic element wherein SXP/SXP-1 ⁇ recombinant antigen is immobilized within the reaction zone; d) Placing the chromatographic element in (c) into the microwell containing the reconstituted mouse monoclonal anti-human IgG4 antibody conjugated to a detection reagent in (a) ; e) Allowing the mouse monoclonal anti-human IgG4 antibody conjugated to a detection reagent to flow from the microwell to the reaction zone of the chromatographic element; and f)
- a method for rapid detection of lymphatic filariasis in a biological sample comprising the steps of: - a) Adding a biological sample to the sample receiving end of a chromatographic element; b) Allowing the biological sample to flow from the sample receiving end to the reaction zone of the chromatographic element wherein SXP/SXP-1 recombinant antigen is immobilized within the reaction zone; c) Adding a buffer to the reagent releasing end of the chromatographic element to reconstitute the dried mouse monoclonal anti-human IgG4 antibody conjugated to a detection reagent incorporated therein,- d) Allowing the mouse monoclonal anti-human IgG4 antibody conjugated to a detection reagent to flow from the reagent releasing end to the reaction zone of the chromatographic element; and e) Detecting the complex formed in (d) .
- a method for rapid detection of lymphatic filariasis in a biological sample comp.fising the steps of : - a) Adding a biological sample to the sample receiving end of a chromatographic element; b) Allowing the biological sample to floW fr.om the sample receiving end to the reaction zol ⁇ e of the chromatographic element wherein SXP/SXP-1 recombinant antigen is immobilized within the reaction z ⁇ ie; c) Adding a buffer to the reagent releasi ng end of the chromatographic element to reconstitute the dried mouse monoclonal anti-human IgG4 antibody conjugated to a detection reagent incorporated therein; d) Allowing the mouse monoclonal anti-human IgG4 antibody conjugated to a detection reagen-i to flow from the reagent releasing end to the control zone of the chromatographic element wherein anti- mouse IgG antibody is immobilized within the control zone,- e) Allowing the mouse monoclonal
- a diagnostic kit for rapid detection of lymphatic filariasis in a biological sample comprising a detection device wherein the detectioa device comprises a chromatographic ' element wherein the chromatographic, element comprises a sample receiving end, a reaction zone and a control zone characterized in that SXP/SXP-1 recombinant antig'en is immobilized within the reaction zone and dried mouse monoclonal anti-human IgG4 antibody conjugated to a detection reagent is in a separate mi ⁇ rowell .
- a diagnostic kit for rapid detection of lymphatic filariasis in a biological sample corrprising a detection device wherein the detection device comprises a chromatographic element wherein the chromatographic element comprises a sample receiving end, a reagent releasing end, a reaction zone ' and a control zone characterized in that SXP/SXP-L recombinant antigen is immobilized within the reaction zone .
- Figure 1 shows the plasmid map of SXP/SXP-I recombinant gene .
- Figure 2 shows the SDS-PAGE profile of SXP/SXP-I recombinant protein.
- S Figure 3 shows the appearance of tests in a. dipstick and cassette format employing the method for rapid detection of lymphatic filariasis .
- the present invention detects anti-filarial IgGA antibodies in a biological sample based on a specific antibody-antigi : ⁇ binding reaction ' , which comprises a recombinant antigen, expressed by the SXP/SXP-1 . gene binding to the anti- 5 filarial Ig ⁇ 34 antibodies in the biological ' sample.
- SXP/SXP-1 gene is a recombinant gene derived from filarial parasites Brugia malayi, Wu ⁇ hereria bancrofti or Brugia timor ⁇ .
- SXP/SXP-1 gene was ' 0 cloned from Brugia malayi cDMA library by established
- the recomb Lnant gene was recloned into a bacterial expression vector 5 (pPRO ⁇ XHT, Life Technologies) and the His-tagged recombinant gene as shown in DNA sequence ID No . : 1 and Figure 1 was transformed into E. coli TOP 10 host (Invitrogen) . Any other expression vector such as prokaryote, insect or mammalian expression vector may be used in the present invention.
- SDS-PAGE analysis of the SXP/SXP-1 recombinant protein in Figure 2 shows that it has an apparent molecular weight of approximately 30 kDa .
- Western blot analysis of the purified recombinant protein showed that the SXP/SXP-1 recombinant antigen produced was sensitive and specific for detection of lymphatic filariasis.
- IgG4 assay in an ELISA format was then developed using SXP/SXP-1 recombinant antigen and the assay was evaluated using serum samples from individuals infected with various infections and. normal individuals. The results showed that the igG4 assay developed was highly sensitive and specific for detection of Nuchereria bancrofti infection.
- the assay was further developed into a rapid assay that employs the SXP/SXP-1. recombinant antigen and the technique of immuno ⁇ hromatograpihy .
- This • ⁇ technology of lateral flow or immunochr ⁇ matography refers to capillary flow of immunological components through an absorbent membrane to mix and s-ybsequentIy separate the various components.
- the rapid immunochromatography assay for detecting filariasis of the present invention includes a chromatographic element comprising three generally contiguous sections : sample receiving end, reaction zone and control zone.
- Chromatographic eletn& ⁇ t refers to a solid matrix upon which the sample can foe applied and allowed to migrate during the assay procedure .
- the chromatographic element particularly preferred in this invention is an absorbent nitrocellulose membrane.
- Other chromatographic elements that can be used include nylon and/ or mixed esters.
- SXP/SXP-1 recombinant antigen is immobilized within the reaction zone and anti-mouse IgG antibody is immobilized within the control zone.
- dried mouse monoclonal anti-human IgG4 antibody conjugated to a detection reagent is prepared separately in a microwell .
- the dried mouse monoclonal anti-human IgG4 antibody conjugated to a detection reagent is incorporated within the reagent releasing end of the chromatographic element.
- Any substance that is capable of producing a detectable signal can be used as the detection reagent conjugated to the mouse monoclonal anti-human Ig.G4 antibody including colloidal metallic particles such as gold and silver, colloidal non-metal particles such as selenium, tellurium and sulfur and also organic polymer latex particles.
- Detection reagents preferred for use' in the present invention are the visually detectable coloured particles, such as colloidal metals, particularly colloidal gold.
- a buffer is added to reconstitute dried mouse monoclonal • anti-human IgG4 antibody conjugated to colloidal gold
- a biological sample such as blood, serum, plasma, urine or tears is introduced to the sample receiving end of an absorbent nitrocellulose membrane and ⁇ is allowed to migrate laterally via capillary action towards the reaction ⁇ zone of the membrane.
- the anti-filarial IgG4 antibodies present in the sample will bind to the SXP/SXP-1 recombinant antigen immobilized within the reaction zone, forming an antibody-antigen -Complex or immune-complex.
- the absorbent nitrocellulose membrane is placed in the microwell containing the reconstituted mouse monoclonal anti-lruman IgG4 antibody conjugated to colloidal gold.
- the mouse monoclonal anti-human IgG4 antibody conjugated to colloidal gold absorbs through the membrane and migrates to the reaction zone and binds to the antibody-antigen complex formed earlier thus forming a complex which comprises SXP/SXP-1 recombinant antigen, anti-filarial IgG4 antibodies and mouse inonoclonal anti-human IgG4 antibody conjugated to colloidal gold.
- the presence of gold in the complex will result in the appearance of a red-purplish line at the location of the reaction zone indicating the presence of anti- filarial IgG4 antibodies in the sample tested.
- the unbound mouse monoclonal anti-human IgG4 antibody conjugated to colloidal gold from the reaction ⁇ zone will further migrate to the control zone and bind with the anti-mouse IgG antibody incorporated therein, forming a red-purplish line in the control zone.
- This control zone serves as an . internal control to ensure the stability of the gold conjugated reagent.
- a biological sample such as blood, seru ⁇ ti, plasma, urine or tears is firs-t introduced to ti ⁇ e sample receiving end of the absorbent nitrocellulose membrane and is allowed to migrate laterally via capillary, action towards the reaction zone of the membrane.
- the anti-filarial IgG4 antibodies present in the sample will bind to the SXP/SXP-1 recombinant antigen immobilized within the reaction zone, forming an antibody-antigen complex or immunocotnplex.
- a buffer is then introduced to the reagent releasing end to reconstitute dried mouse monoclonal anti-lnuman IgG4 antibody conjugated to colloidal gold incorporated therein.
- the mouse monoclonal anti-human IgG4- antibody conjugated -to colloidal gold migrates from the reagent • releasing end to the reaction zone and binds to the antibody-antigen complex formed earlier thus forming a complex which comprises SXP/SXP-1 recombinant antigen, anti-filarial IgG4 antibodies and mouse monoclonal anti-human IgG4 antibody conjugated to colloidal gold.
- the presence of gold in the complex will result in the appearance of a red-purplish line at the location of the reaction zone indicating the presence of anti- filarial IgG4 antibodies in the sample tested.
- the unbound mouse monoclonal anti-human IgG4 antibody conjugated to colloidal go'ld from the reaction zone will further migrate to the control zone and bind with the anti-mouse IgG antibody, forming a rsd-purplish line in the control zone .
- a biological sample such as blood, serum, plasma, urine or tears is first introduced to the sample receiving end of the absorbent nitrocellulose membrane and is allowed to migrate laterally via capillary action towards the reaction zone of the membrane.
- the anti- filarial IgG4 antibodies present in the sample will bind to the SXP/SXP-1 recombinant antigen immobilized within the reaction zone, forming an antibody-antigen complex or imtnunocomplex.
- a buffer is then introduced to the reagent releasing end to reconstitute dried mouse monoclonal anti-human IgG4 antibody conjugated to colloidal gold incorporated therein.
- the mouse monoclonal anti-human IgG4 antibody conjugated to colloidal gold migrates from the reagent releasing end to the control zone and binds with the anti-mouse IgG antibody, forming a red-purplish line in the control zone.
- the unbound mouse monoclonal anti-human IgG4 antibody conjugated to colloidal gold will further migrate to the reaction zone and binds to the antibody-antigen complex formed earlier thus forming a complex which comprises SXP/SXP-1 recombinant antigen, anti-filarial IgG4 antibodies and mouse monoclonal anti -human IgG4. antibody conjugated to colloidal gold.
- the presence of gold in the complex will result in the appearance of a red-purplish line at the location of the reaction zone indicating the presence of anti- filarial IgG4 antibodies in the sample tested.
- the kit comprises a detection device wherein the detection device contains an absorbent nitrocellulose membrane.
- the nitrocellulose membrane has three zones : a sample receiving end, a reaction zone and a control zone characterized by the SXP/SXP-1 recombinant antigen immobilized within the reaction zone.
- Anti -mouse IgG antibody is immobilized, within the control zone-
- the detection device preferably is in a dipstick format .
- the kit further provides a microwell containing dried mouse monoclonal anti-human IgG4 antibody conjugated to colloidal gold,.
- the kit also comprises of. a buffer wherein the buffer comes in a separate container from the detection device .
- the kit which comprises a detection device wherein the detection device contains an absorbent nitrocellulose membrane.
- the nitrocellulose membrane has four zones; a sample receiving end, a reagent releasing end, a reaction zone and a control zone characterized by the SXP/SXP-1 recombinant antigen immobilized within the reaction zone.
- Mouse monoclonal anti-human IgG4 antibody conjugated to colloidal gold is incorporated within the reagent releasing end and anti-mouse IgQ antibody is immobilized within the control zone of the membrane.
- the detection device may be in a dipstick or cassette format.
- the kit also comprises of a buffer wherein the buffer comes in a separate container from the detection device.
- the sample does not contain anti- filarial IgG4 antibodies specific to SXP/SXP-1 recombinant antigen, the complex SXP/SXP-1 recombinant antigen, anti-filarial IgG4 antibodies and mouse monoclonal anti-human IgG4 antibody conjugated to colloidal gold will not be formed, thus no red- purplish line will be seen in the reaction zone, resulting in the final appearance of only one red- purplish line in the control zone.
- two red-purplish lines on the nitrocellulose membrane denotes a positive test result and o ⁇ e line denotes a negative test result as shown in Figure 3.
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US12/297,325 US20100021926A1 (en) | 2006-04-17 | 2007-04-10 | Method for rapid detection of lymphatic filariasis |
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MYPI20061740A MY144980A (en) | 2006-04-17 | 2006-04-17 | Method for rapid detection of lymphatic filariasis |
MYPI20061740 | 2006-04-17 |
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PCT/MY2007/000021 WO2007120034A1 (en) | 2006-04-17 | 2007-04-10 | Method for rapid detection of lymphatic filariasis |
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Cited By (2)
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US4857453A (en) * | 1987-04-07 | 1989-08-15 | Syntex (U.S.A.) Inc. | Immunoassay device |
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2006
- 2006-04-17 MY MYPI20061740A patent/MY144980A/en unknown
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2007
- 2007-04-10 WO PCT/MY2007/000021 patent/WO2007120034A1/en active Application Filing
- 2007-04-10 US US12/297,325 patent/US20100021926A1/en not_active Abandoned
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN107064496A (en) * | 2017-06-05 | 2017-08-18 | 苏州快捷康生物技术有限公司 | A kind of malachite green colloidal gold immunochromatographimethod rapid detection card and preparation method thereof |
CN110068689A (en) * | 2019-05-23 | 2019-07-30 | 贵州盛世康生物科技有限公司 | A kind of Immunoglobulin IgG4 measurement reagent |
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US20100021926A1 (en) | 2010-01-28 |
MY144980A (en) | 2011-12-15 |
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